Brucella melitensis mutants and methods

ABSTRACT

Certain attenuated mutants of  Brucella , especially  B. melitensis, B. abortus, B. suis  and  B. ovis , when administered to a human or animal trigger a protective immune response such that subsequent challenge with virulent  Brucella  of the same species does not result in disease or results in much less severe symptoms. Functional inactivation of galE, a virB gene or the operon (ORFs 1087-1090) comprising the gene encoding β-hexosaminidase (BMEI1087) and a lytic murein transglycosylase gene (BMEI1088). A specific example of the attenuated galE mutant which produces a protective immune response is  B. melitensis  GR024. The specific example of an inactivated ORF1087-1090 operon is  B. melitensis  GR026; it has an insertion mutation in the promoter region upstream of ORF 1090. Vaccination with live cells of either or both of these mutants results in a T cell response which protects the human or animal against challenge with virulent  B. melitensis . Similar strategies for protective immunity using live attenuated mutants are useful for  B. abortus, B. suis  and  B. ovis  as well.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No.60/741,282, filed Dec. 1, 2005, which application is incorporated byreference herein to the extent there is no inconsistency with thepresent disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant R01AI048490awarded by the National Institutes of Health (NIH/NIAID), Grant AI057153awarded by RCE for Biodefense and Emerging Infectious Diseases ResearchProgram, and Grant 35204-14856 awarded by the United States Departmentof Agriculture. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM LISTINGCOMPACT DISK APPENDIX

The Sequence Listing filed herewith is incorporated by reference.

BACKGROUND OF THE INVENTION

The field of this invention is microbial genetics, especially as relatedto immunogenic compositions comprising attenuated bacterial pathogens orcomponents thereof.

The Brucella species are important zoonotic pathogens affecting a widevariety of mammals. In agriculturally important domestic animals, thesebacteria cause abortion and infertility, and they are of seriouseconomic concern worldwide (5). In humans, Brucella species constitutepotential bio-warfare agents. Brucella species that infect humans causein undulating fever, which if untreated, can manifest as orchitis,osteoarthritis, spondylitis, endocarditis, and neurological disorders(11, 46). Currently there is no vaccine to protect against humanbrucellosis, especially that caused by B. melitensis. Treatment ofbrucellosis requires a prolonged combination of antibiotic therapy andis still problematic because of the potential for relapse.

Identifying Brucella virulence factors has been of great interest inunderstanding Brucella pathogenesis and immune evasion. After entry intomacrophages virulent Brucella cells reside in an acidified vacuole, theBrucella containing vacuole (BCV). The BCV transiently interacts withearly endosomes, followed by VirB-dependent sustained interaction withthe endoplasmic reticulum (7). Thus, the BCV matures into a replicativeniche in a VirB-dependent manner (7, 8). VirB proteins forming the typeIV secretion system (T4SS) constitute important factors for Brucellavirulence and intracellular replication (9, 14, 34). Lipopolysaccharide(LPS) is also an important virulence factor (27). Brucella LPS hasminimal endotoxic effect, blocks complement activation, and protectsagainst bactericidal cationic peptides (28). The O-chain is alsoimportant for the conventional entry of Brucella into macrophagesthrough lipid rafts, a route which avoids fusion of the BCV withlysosomes (33, 37). Cyclic β-1, 2 glucan has been shown as an importantvirulence factor required for intracellular survival of Brucella (3).Although T4SS, cyclic β-1, 2 glucan, and LPS are clearly virulencefactors of Brucella, the attenuated mutants lacking these virulencefactors are either considered not safe or insufficient information isavailable to use them as vaccines for humans. This has necessitatedidentification of additional vaccine targets.

Several genetic loci that are required for Brucella replication in vitrohave been identified (14, 24). In vitro conditions may not adequatelyreflect in vivo infection, and therefore, findings may have little or noin vivo relevance (45). In vivo screening methods have been used toidentify Brucella genes required for survival and persistence (18, 26),however, these previous studies have relied on the conventional approachof determining tissue-specific cell counts (CFU) from multiple animalsat different times, a process that is labor intensive and requires largenumbers of animals. Because infection is a dynamic process and varieswithin individual mice, monitoring disease progression temporally withinthe same mouse provides a more comprehensive picture of pathogenicevents. Further, such real-time analysis may reveal virulencedeterminants responsible for tissue specific replication of bacteriathat would not be revealed using conventional CFU enumeration from liverand spleen.

Bioluminescent imaging of mice allows direct visualization of theinfection process and is highly useful for bacterial pathogenesisstudies (10), because the intensity of bioluminescence stronglycorrelates with the number of bacteria in the infected organs (16, 40).Bioluminescent imaging is useful in analyzing sub-acute and chronicinfections that are often difficult to assess using conventionalapproaches because of uncertain bacterial locations (16, 40).

There is a long felt need in the art for safe and effective vaccinesthat protect humans and animals from infection by the pathogenicBrucella species, especially B. melitensis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides attenuated mutants of Brucella, includingBrucella abortus and Brucella melitensis, which are useful in generatingprotective immunity to infection by virulent Brucella, includingBrucella melitensis and Brucella abortus. In particular, mutants inwhich the galE gene (ORF BMEI0921 or the corresponding gene in otherspecies of Brucella) is inactivated are useful in live vaccineformulations and mutants in which one or more peptidoglycan biosyntheticgenes are functionally inactivated, i.e., the genes encoding the lyticmurein transglycosylase and/or β-hexosaminidase are inactivated, forexample polar mutations in the operon in which these genes areexpressed, with the disruption eliminating all, four or three geneswithin the relevant operon (ORFs BMEI1087-1090 in B. melitensis orcorresponding genes/operon in other species of Brucella) are notfunctionally expressed. The mutations resulting in the attenuatedphenotype due to inactivation of galE can be insertion, substitution ordeletion mutations. With respect to the peptidoglycan related genes, itis not entirely sufficient to eliminate functional expression of onlythe dGTP phosphohydrolase gene to produce a mutant which is attenuatedenough to be a desirable vaccine strain. Where the galE-like mutant ofB. melitensis is used, it is recommended that the genetic backgroundinto which the mutation is introduced is a 16M genetic background.

Also within the scope of the present invention, are attenuated mutantsof other species of Brucella, including Brucella abortus, B. suis, B.ovis, etc, where the functionality of the corresponding gene(s) asdescribed above are destroyed. The coding sequence identified by ORFsBMEI1087-1090 are presented in Tables 5 and 6; see also thecorresponding regions of SEQ ID NO:26-27. In strains and species otherthan B. melitensis 16M, from which the sequence information of Tables 5and 6 is derived, the corresponding genes will have at least 85% orhigher nucleotide sequence identity, thus enabling the generation ofequivalent mutants in these coding sequences. Such mutants, whenadministered as live vaccines, provide an immune response to the cognatespecies of Brucella.

Within the present invention, there is at least one attenuated strain ofBrucella in which there is a mutation which functionally inactivates orprevents expression of at least one of the galE and having at least 85%nucleotide sequence identity to SEQ ID NO:28, the gene encoding lyticmurein transglycosylase and having at least 85% nucleotide sequenceidentity to nucleotides 7908-10817 of SEQ ID NO:26, β-hexosaminidase andhaving at least 85% nucleotide sequence identity with nucleotides6688-7740 of SEQ ID NO:26, or a gene encoding deoxyguanosinetriphosphatetriphosphohydrolase and having at least 85% nucleotide sequence identitywith nucleotides 2138-3346 of SEQ ID NO:27. Also encompassed areimmunogenic compositions for administration to a human or animalcomprising an attenuated strain of the present invention. The bacterialcells in the composition can be killed or live, advantageously alive.

Further embodied within the present invention are immunogeniccompositions comprising live cells of attenuated Brucella cells, and apharmaceutically acceptable carrier. These attenuated Brucella cells canbe deficient in the functional expression of at least one gene selectedfrom the group consisting of galE, lytic murein transglycosylase andβ-hexosaminidase. Such compositions include vaccine compositions for usein humans, sheep, goats, cattle, bison and other susceptible animals. Itis understood that the immunization with one particular species ofBrucella results an immune response primarily to same species asadministered. Thus, for protection against B. melitensis, it is desiredto administer an immunogenic composition comprising at least one liveattenuated B. melitensis mutant, as set forth herein. These compositionscan further comprise an agent which stimulates the immune response, forexample, an interleukin such as interleukin 12.

The present invention further provides methods for generating an immuneresponse, especially a protective immune response in humans, sheep,goats, and other animals. Desirably, the immune response generated is aT cell response. A single injection with live cells of least oneattenuated Brucella mutant strain (desirably from 10³ to 10⁸ viablecells of each strain) as set forth above can trigger a protective immuneresponse in the human or animal to which it has been administered, dueto the persistence of the bacterial within the body of that animal. Theimmunogenic composition can be administered by any route ofadministration, such as subcutaneous, intramuscular, intraperitoneal,intravenous, mucosal, intradermal and so on. Because of the ability ofthe attenuated mutants of the present invention to persist in the body,it is not necessary for there to be repeated administrations of theimmunogenic composition, although booster immunizations may be given.

Also within the scope of the present invention are attenuated mutants ofBrucella strains having the same or equivalent defects to those of GR024and GR026, as described herein, in which the hly gene (listeriolysin O)of Listeria monocytogenes is expressed. This results inbrucella-infected cells which are “leaky”, thus resulting in a moreeffective immune response.

The present invention further provides methods for identifying B.melitensis peptides that correspond to MHC class I-restricted T cellepitopes, especially those associated with MHC class I (H-2K^(d)).

Additionally, the present invention provides a number of peptides thatare associated with intracellular survival strategies of Brucella. Theseinclude several derived from an extracellular serine protease(BMEEII0148), characterized by a carboxy terminal region (amino acids2349-2554) with high sequence homology to the β-domains ofautotransporters of the Type V Secretory Systems of bacterial pathogens.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a real-time analysis of the attenuatedbioluminescent B. melitensis strains in IRF-1^(−/−) mice. IRF-1^(−/−)mice were infected with 1×10⁷ CFU of B. melitensis strains GR019(virB4), GR024 (galE) and GR026 (90-91IR) and imaged daily with a 10 minexposure. Numbers at the bottom indicate days PI. Unlike GR019,infection with GR024 or GR026 resulted in a localized bioluminescencesuggesting a defect in systemic spread. Rainbow scale representsapproximate photon counts.

FIGS. 2A-2B illustrate replication kinetics of bioluminescent B.melitensis strains. FIG. 2A: Stationary phase grown cultures (30 μl)were inoculated into 30 ml of brucella broth and grown at 37° C. withshaking and OD₆₀₀ was determined. FIG. 2B: RAW264.7 macrophages wereinoculated with a standardized bacterial suspension of different strainsand growth monitored at specified times. The CFU counts were logtransformed and values are average ±standard error for duplicatesamples.

FIG. 3 provides a schematic representation of EZ::TN<lux> transposoninsertion in the three attenuated bioluminescent B. melitensis strains(only relevant features are shown; picture not drawn to scale).EZ::TN<lux> transposon is indicated as a closed hexagon relative to thesite of insertion. The relevant ORFs upstream and downstream of theinsertion are shown in open boxes with arrows indicating direction oftranscription with numbers corresponding to the B. melitensis 16M genomesequence. The orientation of the arrow below the transposon in eachstrain represents the direction of Lux expression based on our sequencedata. The sites for ClaI restriction enzyme used in Southernhybridization experiment are shown by the letter C.

FIG. 4A illustrates complementation of GR019 with the virB operon fullyrestored growth in macrophages. FIGS. 3B and 3C show virulence inIRF-1^(−/−) mice. RAW264.7 macrophages were inoculated with astandardized bacterial suspension of GR019 and GR019 complementedstrains, and the growth was monitored at specified times. The CFU countswere log transformed and values are average ±standard error forduplicate samples. IRF-1^(−/−) mice were inoculated i.p. with 1×10⁷ CFUof GR019 and GR019 complemented strains, and mouse survival (FIG. 34) aswell as CFU from livers, spleens and testes (FIG. 4C) were determined.The CFU counts from livers, spleen and testes were log transformed andthe data are an average of 4 mice. Error bars represent the range ofCFU.

FIG. 5 demonstrates that GR024 and GR026 protect IRF-1^(−/−) mice fromchallenge with virulent B. melitensis. IRF-1^(−/−) mice (n=9) immunizedwith different attenuated B. melitensis strains (1×10⁷ i.p./mouse) werechallenged with virulent B. melitensis GR023 (1×10⁶) and monitored forsurvival.

FIG. 6A shows the results of bioluminescent monitoring of virulent B.melitensis infection in vaccinated IRF-1^(−/−) mice. IRF-1^(−/−) micevaccinated with different attenuated strains were imaged for 10 minfollowing GR023 challenge. Numbers at the bottom of each figure indicatedays PI and images representing same PI day from different groups areshown. Rainbow scale represents approximate photon counts. FIG. 6B showsthe results of bioluminescent imaging of surviving IRF-1^(−/−) micefollowing challenge (upper panel) and the corresponding histologicalchanges in livers and spleen (lower panel). Livers were scored by thenumber of focal granulomas observed per field of view (fov) at 4×magnification. Data represent the average number of granulomas from 8fov. (+) 1-8; (++) 9-16; (+++) 17-24 granulomas. Spleens were scored onloss of white and red pulp architecture at 4× magnification; (−) normalspleen or no noticeable changes, (+) enlarged follicles, increasedcellularity, and white pulp, (++) hyperplasia, with a significantincrease in follicle size, and white pulp. (+++); increased red pulp andloss of white pulp architecture.

FIG. 7A illustrates real-time analysis of attenuated bioluminescent B.melitensis strains in C57BU6 mice. C57BU6 mice were infected with 5×10⁷cfu of B. melitensis strains GR019, GR024, and GR026 and imaged dailywith a 10 min exposure. Numbers at the bottom indicate days PI andimages representing same PI day from different groups are shown. Similarto IRF-1-1^(−/−) mice, GR024 and GR026 resulted in only a localizedbioluminescence suggesting a defect in systemic spread. Rainbow scalerepresents approximate photon counts. FIG. 7B shows the results ofbioluminescent monitoring of the virulent B. melitensis infection invaccinated C57BL/6 mice. C57BL/6 mice vaccinated with differentattenuated strains were challenged with GR023 and imaged for 10 min.Rainbow scale represents approximate photon counts.

FIGS. 8A-8B shows the results of CFU counts from livers and spleens,respectively, of C57BU6 mice vaccinated with different attenuatedstrains followed by virulent GR023 challenge. The CFU counts were logtransformed and the data are an average from 3-4 mice at each timepoint. Error bars represent the range of CFU of the samples from eachtime point.

FIG. 9A is a photomicrograph of large grossly visible focal calcifiedgranulomas in C56BL/6 mice vaccinated with Rev-1. FIGS. 9B-9C arephotomicrographs of large focal granulomas contained secondary changesincluding a central area of necrosis, neutrophil infiltration, andfibrosis with calcification.

FIG. 10 illustrates the generation of antigenic peptide MHC class Icomplexes. As shown in the control experiment, a recombinant E. coliexpressing the Yersinia enterocolitica invasin (inv) and the Listeriamonocytogenes listeriolysin O (Hly) as well as a green fluorescentprotein (GFPuv) was allowed to infect macrophages in culture for 24 hrs.

FIG. 11 shows the strategy for the identification of relevant peptideepitopes of B. melitensis. Brucella infects macrophages in culture; MHCclass I proteins complexed with epitopic peptides are purified by MHCI-specific immunoaffinity chromatography, the peptides are eluted andthen those peptides are characterized by mass spectroscopy. The invasivelisteriolysin-expressing E. coli are transformed with plasmidsexpressing the peptide epitopes identified; these cells are useful forimmunizing humans or animals with Brucella-specific peptides.

FIG. 12 provides schematic illustrations of the Brucella extracellularserine protease (encoded by ORF BMEII0148), which contains β-domain(amino acids 2349-24554) and passenger domain (amino acids 1-2348). Thisstructure is characteristic of autotransporters of Type V secretoryproteins. The peptide of the Brucella extracellular serine protease wasisolated from MHC class 1 molecules following a 24 hr infection ofmacrophage cells in culture; the peptide was identified by MALDI-TOFmass spectroscopy.

FIG. 13 summarizes the results of the demonstration project in which theInv+ Hly+ E. coli expressing the GFPuv protein was cultured inmacrophages. The MHC class I-peptide complexes were collected, thepeptides were eluted and then characterized. Within the peptidesisolated was NYNSHNVYIT (SEQ ID NO:25) from within the GFPuv protein.

FIGS. 14A-14B summarizes the experiment carried out with the invasiveInv+ Hly+ E. coli transformed with the B. melitensis hdeA (smallchaperone protein functioning in Type II secretion) gene and the GFPuv(a green fluorescent protein) gene. These E. coli strains were injectedinto mice intraperitoneally. The invasive E. coli generatesantigen-specific cytotoxic T lymphocytes in the mice. After 6 wks,cytotoxic lymphocyte (CTL) assays were performed with transduced targetcells. FIG. 14A shows the results of the CTL assay carried out withserum from mice immunized with the Inv+ Hly+ E. coli expressing theGFPuv protein. FIG. 14B shows the results of the CTL assay carried outwith serum from a mouse immunized with the Inv+ Hly+ E. coli expressingthe B. melitensis HdeA protein.

FIG. 15 shows the bioluminescence transposon used to produce the GR019,GR024 and GR026 mutants described herein. GR023 is an insertion mutantwhich was not affected with respect to virulence in the mouse model. Thestrategy used for analyzing the bioluminescence transposon insertionmutants is also shown.

DETAILED DESCRIPTION OF THE INVENTION

Certain bioluminescent mutants of B. melitensis are avirulent inIRF-1^(−/−) mice. IRF-1^(−/−) mice are highly susceptible and succumb tovirulent Brucella infection; however, their response varies with thevirulence of the Brucella strains (21, 22). Therefore, attenuatedstrains can be readily identified using these mice. We tested the threeEZ::TN/lux bioluminescent mutants, GR019, GR024 and GR026 in IRF-1^(−/−)mice to determine the virulence and pathology associated with thesestrains. In addition, we also tested two other B. melitensis mutants,BM710, a rough strain and Rev-1, a vaccine strain, so that thebioluminescent mutants could be evaluated for their ability to conferprotection against challenge with virulent B. melitensis. IRF-1^(−/−)mice (n=4) infected with bioluminescent strains were monitored forbacterial dissemination and persistence. Bioluminescence spreadsystemically in GR019 infected mice by day 1 post infection (PI),however, in the GR024 or GR026 infected mice bioluminescence localizedprimarily at the injection site (FIG. 1). By day 2 strongerbioluminescence was observed in many areas including the submandibularregion only in GR019 infected mice. However, by day 6 GR019 infectedmice began to clear the infection indicated by reduced bioluminescenceand by day 24 minimal bioluminescence was observed in the extremities(FIG. 1). In contrast, in both GR024 and GR026 infected micebioluminescence was predominantly observed at the injection site.However, by day 12, bioluminescence began to appear in the tail asmultifocal lesions and was more prominent in GR024 infected mice by day24 (FIG. 1). Mice infected with all three bioluminescent strainsappeared healthy and survived longer than 24 days suggesting attenuationof these strains. Similarly mice infected with rough B. melitensisstrain BM710 survived greater than 24 days suggesting attenuation.However, all Rev-1 infected mice died by 7 days PI. Although Rev-1 is acommercial vaccine, it was fully virulent in these mice. To determinethe relative pathology associated with different attenuated mutants, thelivers and spleens were processed for CFU and histopathology. Livers andspleens from GR019 or BM710 infected mice had lower CFU counts comparedto GR024 or GR026 infected mice (Table 2). However, except for the GR026infected group, livers and spleens from other groups had no observablehistological changes (Table 2, data not shown). GR026 infected micedisplayed very few multi-focal granulomas in livers and minor changes inthe white pulp of spleens.

GR019, but not GR024 or GR026, is attenuated in RAW macrophages. Weexamined the growth of bioluminescent mutants in RAW macrophage-likecells. All three insertion mutant strains exhibited growth rates similarto that of the virulent parent strain 16M with a duplication time of 2hr in brucella broth suggesting no general growth defects in GR019,GR024 and GR026 (FIG. 2A). RAW macrophages were infected with eachstrain at a MOI of 1:50 and the growth was monitored for 72 hrs.Interestingly, only GR019 was defective in replication with asignificant decrease in intracellular bacteria by 24 hr PI compared to16M (FIG. 2B). On the other hand, both GR024 and GR026 displayed agrowth distinct from GR019 or 16M. Both strains were phagocytosed morewith no apparent intra-macrophage replication during 24 hr PI asbacterial levels remained constant. However, by 48 hr their growth wassimilar to 16M. The growth curves of GR024 and GR026 appeared asintermediate between a virulent (e.g., 16M) and rough (e.g., perA, GI-2mutant) strains of Brucella (41, unpublished data). Rough strains ofBrucella are phagocytosed at even higher number and are defective inintra-macrophagic replication though they persist in higher numbers formore than 3 days in culture (35, 20, and unpublished data). This led usto suspect that both GR024 and GR026 are defective in LPS. Therefore wetested the LPS phenotype of these two mutants by the acriflavinagglutination test (6). As suggested by the macrophage growth pattern,both strains resulted in fine amorphous agglutination particles thatwere less intense compared to known LPS rough strains of Brucella (datanot shown). In addition, unlike GalE mutants from other bacteria, GR024did not show any sensitivity to galactose (1, 36, data not shown).

To determine the EZ::TN/lux insertion site in GR019, GR024, GR026, weperformed ‘rescue cloning’ of the R6KΔori present in EZ::TN transposonfrom genomic DNA of each strain. Nucleotide sequencing of the rescuedR6K plasmid clones in both orientations identified the transposoninsertion in VirB4 (BMEII0028) for GR019, GalE homolog (BMEI0921) forGR024, and in the intergenic region of BMEI1090-1091 for GR026 (FIG. 3).The type IV secretion system encoded by 11 ORFs virB1-11 are transcribedas a polycistronic message and the disruption of these genes has beenshown to attenuate Brucella in macrophages as well as in mice (9, 14,34). Similarly, in GR024, the insertion disrupted the GalE homolog(BMEI0921) that has been previously shown to attenuate Brucella (38).However, in GR026 the insertion was located in the intergenic region oftwo divergent ORFs BMEI1090 and BMEI1091. Annotation of the B.melitensis genome suggested that BMEI1090 is the first gene in a clusterof genes that are transcribed in minus orientation, whereas BMEI1091 isan independent transcriptional unit (FIG. 3). Based on the sequence dataobtained by transposon mapping, we have concluded that the alteredexpression of BMEI1090 or its downstream genes is responsible forattenuation of GR026. Further, Southern blot analysis confirmed thesequencing results and also revealed the single copy insertion of thetransposon in these strains.

To determine the gene(s) likely responsible for the observed phenotypeof GR026, we created non-polar mutations in BMEI1090 and 1091 by allelicreplacement. The respective ORFs were replaced with a kan^(r) marker byhomologous recombination and resulting strains, GR-1090Δ and GR-1091Δ,were tested for virulence in IRF-1^(−/−) mice. IRF-1^(−/−) mice infectedwith GR-1091Δ died within 10 days similar to virulent 16M; however, onlytwo mice infected with GR-1090Δ died and the remaining mice survived forat least 21 days (Table 2). The livers and spleens from the survivingmice had an average CFU of 6.65E+04 and 1.14E+06, respectively.Therefore inactivation of 1090 resulted in partial attenuationsuggesting the phenotype associated with GR026 is likely due to alteredexpression of I1090 and its downstream genes.

To confirm that the attenuation of bioluminescent mutants is due todisruption of transposon insertion targets and not due to secondarymutations, we complemented GR019, GR024 and GR026 with the correspondingORFs. Because GR019 has a growth defect in RAW macrophages, the GR019containing either pBBVirB4 or pBBVirB were tested for growth in thesemacrophages. Introduction of pBBVirB4 into GR019 resulted in partialrestoration of the ability to grow in macrophages, as reflected byincrease in intracellular bacteria at 24 hr Pi (FIG. 4A). However,addition of pBBVIrB, containing the entire virB operon (34), into GR019resulted in complete restoration of growth (FIG. 4). In addition, GR019complemented with pBBVirB but not pBBVirB4, was able to kill IRF-1^(−/−)mice and restore complete virulence (FIG. 4B). Consistent with the invitro results, mice infected with pBBVirB4-complemented GR019 did notdie and contained more bacteria in livers and spleens compared to GR019infected group (FIG. 4C).

Because both GR024 and GR026 agglutinated in the presence of acriflavin,we tested the GR024 and GR026 complemented strains for agglutination.GR024 complemented with pBBGalE resulted in no agglutination in thepresence of acriflavin. Our earlier results suggested that the observedphenotype for GR026 is due to the altered expression of 11090 and itsdownstream genes, so we complemented GR026 with a plasmid containing 4ORFs likely to form an operon (12). Surprisingly, addition of pBB1087-90to GR026 resulted in much more pronounced agglutination, as seen withrough strains of Brucella. The functions encoded by BMEY1087-1090 areα-hexosamionidase, soluble lytic murein transglycosylase, arginyl tRNAsynthetase and deoxyguanosinetriphosphate triphosphohydrolase.Consistent with the acriflavin agglutination results, both GR024 andGR026 were partially resistant to smooth-type specific Tbilisi (Tb)phage, and the addition of pBBGalE restored the susceptibility of GR024to Tb phage. However, GR026 complemented with pBBI1087-90 was completelyresistant to Tb phage suggesting a rough phenotype of the complementedstrain.

GR024 and GR026 protect IRF-1^(−/−) mice from virulent challenge.IRF-1^(−/−) mice, though immuno-compromised, have been shown to generatea protective immune response following vaccination with attenuatedstrains (22). Therefore, we tested the abilities of the attenuatedbioluminescent mutants to protect IRF-1^(−/−) mice from virulentchallenge. IRF-1^(−/−) mice (n=9) were vaccinated by intraperitonealinjection with 1×10⁷ CFU of each Brucella strain, and 60 days aftervaccination, the mice were challenged with 1×10⁶ CFU of virulentbioluminescent B. melitensis strain GR023 (40). IRF-1^(−/−) micevaccinated with attenuated bioluminescent mutants were challenged byintraperitoneal injection when no bioluminescent bacteria weredetectable. The GR023 strain of B. melitensis was used for challengestudies to evaluate vaccine candidates for the ability to alter thedissemination and localization of virulent Brucella to different tissuesas visualized temporally in individual mice by imaging. All micevaccinated with either GR024 or GR026 survived for at least 44 days,where as only 2 mice vaccinated with GR019 and 3 mice vaccinated withBM710 survived for 44 days following challenge (FIG. 5). Fifty percentof GR019 vaccinated mice died by day 12, whereas 50 percent of the BM710vaccinated mice died by day 9 following challenge. As expected, all theunvaccinated mice died within 2 weeks following challenge with fiftypercent of mice being dead by 7 days (FIG. 5).

The livers and spleens from surviving mice vaccinated with differentstrains had very similar CFUs (CFU ranges; liver: 2.2E+02-1.2E+03,spleen: 1.5E+04 to 3.4E+04). Bacteria recovered from livers and spleensof mice vaccinated with bioluminescent strains were confirmed as theGR023 challenge strain by verifying the insertion site of EZ::TN<lux>using PCR. Bioluminescent imaging of vaccinated mice following i.p.challenge revealed strikingly different dynamics of persistence andspread of virulent bacteria. Unlike the unvaccinated mice, in allvaccinated groups, bacterial spread was less extensive (See FIGS. 6A and6B), but correlated with ability of the vaccine strain to protect fromchallenge. In both BM710 and GR019 vaccinated groups, bioluminescencewas pronounced with systemic spread; however, in both GR024 and GR026groups, bioluminescence was observed at the site of injection and in thetail region (see FIGS. 6A-6B). By day 44 both GR024 and GR026 vaccinatedmice had no detectable bioluminescent bacteria while both BM710 andGR019 vaccinated survivors still exhibited detectable bioluminescence(FIG. 6B). Consistent with IRF-1^(−/−) mice survival data, the GR024 andGR026 vaccinated mice had the least histological changes in livers andspleens. The GR024 and GR026 vaccinated mice had only few focalgranulomas (less than 3/field of view) in the liver sections, while thespleens of GR024 vaccinated mice appeared normal with only minimaldisorganization of the splenic white pulp in GR026 vaccinated mice.However, both GR019 and BM710 vaccinated survivors had more histologicalchanges in both livers and spleens compared to GR024 or GR026 groups(see FIG. 6B).

IRF-1^(−/−) mice are defective in multiple aspects of the immune system(44). Therefore, to better correlate the immune protection provided bythe different attenuated strains, we tested these bacterial strains inwild type C57BU6 mice, the parental strain of IRF-1^(−/−) mice. C57BU6mice are susceptible to virulent Brucella infection naturally and serveas a relevant model in which to study Brucella pathogenesis and immuneprotection. To assess the protection by different attenuated strains, wemonitored bacterial clearance and histological changes in livers andspleens. In addition, the dynamics of infection by attenuatedbioluminescent strains and their effects on virulent challenge weremonitored by imaging. Similar to IRF-1^(−/−) mice, GR019 vaccinatedC57BU6 mice had bioluminescence in systemic organs by day 1 PI; however,in GR024 or GR026 vaccinated mice bioluminescence was detected primarilyat the injection site (FIG. 7A). Bioluminescence began to diminish byday 5 in all groups and by 2 weeks PI minimal or no bioluminescence wasobserved (FIG. 7A). However, after challenge the dynamics of virulentBrucella spread was similar in all vaccinated groups being limitedprimarily to the injection site, although bioluminescence was strongerin GR019 and BM710 vaccinated groups (FIG. 7B). Consistent with imagedata, all vaccinated groups had at least 2 logs less CFUs from liversand spleens at 1 week post challenge with Rev-1 and GR024 vaccinatedgroups containing even lower numbers of CFUs (FIG. 8). Similarly, at 2weeks post challenge, livers from Rev-1 and GR024 vaccinated groups hadsignificantly lower CFU compared to other groups. However, spleens fromGR024 and Rev-1 vaccinated mice had lower CFU at all times compared toother groups though Rev-1 vaccinated mice had significantly fewer CFUcompared to other groups (FIG. 8). To correlate the bacterial clearancewith the tissue damage, histological changes were assessed in livers andspleens from immunized mice following challenge. Consistent with thebacterial clearance, GR024 and Rev-1 vaccinated mice exhibited fewergranulomas in liver at all times; however, livers from GR019 and BM710vaccinated mice contained more granulomas (Table 3). Surprisingly, thelivers from all Rev-1 vaccinated mice had large grossly visible focalcalcified granulomas (FIG. 9). On the other hand, histological changesin spleens were similar in all vaccinated groups but contained fewerchanges compared to unvaccinated controls (Table 3).

Mice are used extensively to study Brucella pathogenesis; however, theinterpretation of data is often limited to CFU or histological changesobserved in specific tissues. These approaches have limited ourunderstanding of the dynamics of Brucella dissemination and localizationinto tissues beyond those organs that are conventionally used forevaluation. In this report, we describe the infection dynamics of threeattenuated bioluminescent mutants in mice by visualizing how infectiondisseminates, bacterial preference to organs, contribution of certainBrucella genes to pathogenesis, and effect of vaccination on thedynamics of virulent bacterial infection. GR019, GR024, GR026, and BM710were all attenuated in IRF-1^(−/−) mice; however, Rev-1 remainedvirulent in these mice. Imaging of mice infected with bioluminescentstrains revealed striking differences in bacterial dissemination andpersistence. GR019 (VirB4), unlike GR024 or GR026, spread systemicallyand bioluminescence was observed in liver, spleen, testes, submandibularregion and extremities early in infection, suggesting that the VirBsystem is not important for establishing early infection. However, theVirB system is required for Brucella persistence because C57BU6 micecleared GR019 infection faster than virulent Brucella. GR024 (GalE) andGR026 (90-911R), on the other hand, failed to disseminate systemically(FIG. 1). Interestingly, in both GR024 and GR026 infected mice, signalsreappeared 12 days PI and localized in the joint-rich tail region duringthe later stages of infection (FIG. 1), suggesting that virulence isregulated differently in GR024 and GR026. Bioluminescent imaging iscritical in identifying the contribution of Brucella genes topreferential tissue localization of Brucella. In addition, temporalbioluminescence analysis of infection revealed patterns of growth andclearance, as well as reemergence of bacteria, that is extremelydifficult to observe with conventional methods. Thus, our study clearlydemonstrates that conventional CFU enumeration is useful but not idealto assess Brucella clearance. Importantly, only GR019 was attenuated invitro in RAW macrophages (FIG. 2B). Therefore, in vivo imaging mayprovide a more comprehensive approach to identify Brucella virulencegenes that are relevant to in vivo pathogenesis. Although GR024 andGR026 localized in the tail region in later stages of infection, no(GR024) or very minimal (GR026) histological changes in livers andspleens were observed, similar to GR019 or BM710 infected groups. Thus,these strains, individually or mixed together in combinations of two orthree mutants, or two or three mutations in a single strain, are usefulin the formulation of immunogenic compositions, including vaccines.candidates and bioluminescent imaging may be highly useful for vaccineselection.

Both GR024 and GR026 exhibited growth patterns in macrophagesintermediate between those of smooth and rough strains of Brucella (41),and both strains produced very fine agglutination particles in thepresence of acriflavin and were partially resistant to smooth-typespecific Tb phage, suggesting that they have an altered surfacestructure (30). In GR024, the transposon insertion is in ORF BMEI0921, aNAD dependent epimerase/dehydratase family member that is closelyrelated to enterobacterial galE. The galE gene is an important virulencefactor in many Gram negative bacteria and is involved several cellularprocesses including cell membrane biogenesis (15, 17, 29, 32, 39, 42).The galE mutants in other bacteria possess defective LPS, reflecting acontribution of galE to LPS biogenesis. Likewise, acriflavinagglutination and phage susceptibility tests suggest a defect in theGR024LPS; however, GR024 was not sensitive to galactose. The galEmutants of other bacteria display a variable response to galactose, withsome being sensitive while others are not sensitive to galactose (15,19, 39). The B. melitensis genome contains another member of the NADdependent epimerase/dehydratase family, BMEII0730. BMEII0730 is moreclosely related to UDP-glucose 4-epimerases from members of theα-proteobacteria and shares no homology with BMEI0921. A few bacterialspecies have two functional galE genes. In Yersinia enterocolitica onegalE gene is linked to galactose utilization genes and the other linkedto the LPS synthesis genes (39). However, neither of the Brucella galEgenes is linked to galactose metabolic genes or to LPS biosyntheticgenes. Although our results indicate that BMEI0921 plays a role in cellmembrane biogenesis, whether it is involved in galactose utilization isnot clear because the growth of GR024 was not inhibited ingalactose-containing medium. Brucella genome annotation suggest thatBrucella BMEI10730 is linked to sugar metabolism genes and may beinvolved in galactose utilization.

GR026 has an insertion in the intergenic region between BMEI1090 and1091. Further, selective allelic replacement of BMEI1090 or BMEI10191supported the conclusion that loss of function of BMEI1090 and itsdownstream genes is responsible for the attenuation of GR026 (Table 1).BMEI1090 or BMEI10191 encode HesB protein and a theoretical protein,respectively. Without wishing to be bound by any particular theory, wehave concluded that 1090 and its downstream genes (1087-1090) form anoperon. BMEI1087 encodes α-hexosaminidase A, while BMEI1088 encodessoluble lytic murein transglycosylase, and these are involved in aminosugar metabolism and N-glycan biosynthesis (kegg database). Therefore,this operon may contribute to cell membrane and/or wall biogenesis.Consistent with this observation the acriflavin agglutination and Tbphage susceptibility tests suggested that GR026 has a surface structuredefect. Complementation of GR026 with a plasmid containing BMEI1087-1090ORFs resulted in more pronounced agglutination and complete resistanceto Tb phage suggesting that the expression of these genes are understrict regulation.

Both GR024 and GR026 protected IRF-1^(−/−) mice from virulent B.melitensis challenge, whereas highly attenuated GR019 and BM710 failedto protect these mice. In addition, GR024 and GR026 vaccinated micedisplayed minimal changes in livers and spleens and no bioluminescencewas observed at 44 days post-challenge. IRF-1^(−/−) mice are defectivein multiple immune components with reduced numbers of CD8⁺ T cells,functionally impaired natural killer cells, and dis-regulation of IL-12p40 and inducible nitric oxide synthase (44). Though these mice areseverely immuno-compromised, they mount an adaptive immune responsesufficient to protect against virulent challenge and protection isvaccine strain dependent. Unlike, GR019, both GR024 and GR026 produced alocalized but persistent infection in these mice (FIG. 1) and induced aprotective immune response against virulent Brucella that may requiresome persistence of the vaccine strain. Similar results have beenobserved with two field vaccines stains, S19 and RB51 (23, 43). S19persist longer and is more protective than RB51 in mice and other models(23, 43). However, S19 still possess residual virulence in domesticanimals and in IRF-1^(−/−) mice (22, 31), whereas RB51 is highlyattenuated (22). GR024 and GR026 are highly attenuated in IRF-1^(−/−)mice similar to RB51; however, they cause no or very minimalpathological changes in livers and spleen and are protective. Consistentwith the IRF-1^(−/−) mice data, both GR024 and GR026 provided greaterprotection to C57BLU6 mice than GR019 or BM 710 suggesting thatIRF-1^(−/−) mice may serve as an important model to rapidly assessvaccine efficacy of Brucella strains. Interestingly Rev-1 vaccinatedmice had fewer CFU in both livers and spleens compared GR024 or GR026vaccinated mice; however, Rev-1 vaccinated mice displayed severe liverdamage with grossly visible lesions (FIG. 9) that was not seen in othergroups. These lesions are likely vaccine induced as they were apparenteven at 1 week post challenge. Rev-1 vaccine is used in domestic animalswhere B. melitensis is endemic with varying degrees of success (4).Although Rev-1 protected wild type mice, Rev-1 was highly virulent toIRF-1^(−/−) mice (Table 2) and caused severe liver damage in wild typemice. In summary, our study revealed contribution of Brucella genes toin vivo pathogenesis and identified a new set of virulence genes(BMEI1090 and its downstream genes). Further, the galE deficient GR024has altered LPS structure, results in no or very minimal tissue damage,and protects against virulent B. melitensis challenge making it aninteresting vaccine candidate for brucellosis.

While the immunization strategy has been described using particularmutants of B. melitensis it is understood that corresponding mutants canbe made in other species of Brucella, for use in immunogeniccompositions and vaccination strategies for protection of the cognatespecies of Brucella. It is understood that there may be someimmunological cross reactivity between species of Brucella, the mosteffective protection is afforded by immunization with an attenuatedmutant of the same species as that for which protection is sought.

Further to the particular insertion and deletion mutants or those havingequivalent loss of function as GR024 and GR026 described herein,immunogenic compositions and vaccines can be prepared using such mutantsin which the listeriolysin (hly) derived from Listeda monocytogenes isexpressed. Expression of this protein results in phagosomes which are“leaky”. The intracellular bacteria from the phagosomes are releasedinto the cytoplasm of the cells in which they are reproducing, and thereis a better immune response triggered. See, for example, Grode et al.(2005) J. Clin. Invest. 115: 2472-2479. For further discussions oflisteriolysin, see also Giammerini et al. 2003. Protein Expr. Purif.28:78-85; Dancz et al. 2002. J. Bacteriol. 184:5935-5945, Mengaud et al.1988. Infect. Immun. 56:766-772 among others.

We have identified and analyzed B. melitensis-specific MHC classI-restricted T cell epitopes. There is additional data of MALDI-TOF Massspectral analysis of such peptides naturally processed and associatedwith MHC class 1 molecules from macrophages infected with Brucella for24 hrs. We have identified over 2500 peptides identified as Brucellaassociated with MHC class I (2 K^(d)). These include peptides derivedfrom the ORFs, as identified in Table 4.

Analysis of the peptides associated with MHC class I (2 K^(d)) hasrevealed that a number of the peptides are likely associated withproteins previously unknown to be a part of Brucella's intracellularsurvival strategies. For example, one of the identified peptides is froman extracellular serine protease (BME10148). This protein has aconserved β-domain at the carboxy-terminal region that has high sequencehomology to the β-domains of autotransporters of the Type V secretorysystem of bacterial pathogens (see FIG. 10). Without wishing to be boundby theory, we believe that Brucella uses this Type V secretory systemprotein as an intracellular survival or virulence strategy inmacrophages. Peptide epitopes identified by this strategy can beexpressed by nonreplicating, nonpathogenic E. coli cells which have beengenetically modified to express the Yersinia enterocolitica inv gene andthe hly gene from Listeria monocytogenes. The invasin confers theability to invade nonprofessional phagocytic cells. Binding of invasionto β1 integrin expressed on mammalian cells is necessary and sufficientto induce phagocytosis of the bacteria. After internalization, E. coliis taken into the phagosome/lysosome where lysis of the bacteriumoccurs. Among the various bacterial proteins released into the lysosomalvesicle, listeriolysin present in the cytoplasm of the invasive E. coligains access to the phagosomal membrane, perforating it a low pH. Thecytoplasmic contents of the bacteria can then escape into the cytosoliccompartment of the mammalian cell through the pores generated bylisteriolysin. Using this mechanism, it was demonstrated that invasiveE. coli can be used as a delivery vector for therapeutic proteins.Furthermore, invasive E. coli can elicit a specific CTL response andthus, expression of Brucella proteins or peptides which can elicit aneffective and protective T cell response within a mammalian cell andrelease from the phagosome via listeriolysin provides for a usefulvaccine against brucella infections. See FIG. 13A and FIG. 13B for theresults of CD8 CTL assays carried out with serum from mice immunized byintraperitoneal injection with live Inv+Hly+E. coli expressing the GFPuvprotein or the HdeA protein of B. melitensis. Such vaccine(s) arevaluable for protection of humans and animals against the cognatespecies of Brucella; human welfare and improved animal health, withbenefits to agriculture, animals in captivity and the like are achieved.

As used herein, attenuated means that a bacterial strain is reduced invirulence as compared to a “wild-type” clinical strain that causesdisease in a human or particular animal; the attenuated strain does notcause disease in the human or particular animal.

With reference to a mutation, functional inactivation of a gene meansthat there is little or no activity of the gene product. For example,where the gene encodes an enzyme, the encoded product has less than 10%,desirably less than 5% or less than 1% of the enzymatic activity of theproduct from the wild type gene or there is less than 10%, less than 5%or less than 1% of the expression product. That is to say that thecoding sequence can be interrupted with an inserted nucleotide orsequence, partly or wholly deleted or there can be a substitutionmutation that changes the amino acid sequence of the encoded proteinsuch that activity is significantly reduced. Alternatively, there can bean insertion, deletion or change in transcription and/or translationregulatory sequences such that expression is reduced or prevented at thelevel of gene transcription and/or translation of mRNA.

When a compound is claimed, it should be understood that compounds knownin the art including the compounds disclosed in the references disclosedherein are not intended to be included. When a Markush group or othergrouping is used herein, all individual members of the group and allcombinations and subcombinations possible of the group are intended tobe individually included in the disclosure.

Every formulation or combination of components described or exemplifiedcan be used to practice the invention, unless otherwise stated. Specificnames of compounds and/or genes or mutants are intended to be exemplary,as it is known that one of ordinary skill in the art can name the samecompounds differently. One of ordinary skill in the art will appreciatethat methods, starting materials, mutagenic methods, compositions,vaccine regiments and immunogenic composition ingredients other thanthose specifically exemplified can be employed in the practice of theinvention without resort to undue experimentation. All art-knownfunctional equivalents, of any such methods, starting materials, geneticmethods, and formulations and vaccination regiments are intended to beincluded in this invention. Whenever a range is given in thespecification, for example, a temperature range, a time range, or acomposition range, all intermediate ranges and subranges, as well as allindividual values included in the ranges given are intended to beincluded in the disclosure.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. Any recitation hereinof the term “comprising”, particularly in a description of components ofa composition or in a description of elements of a device, is understoodto encompass those compositions and methods consisting essentially ofand consisting of the recited components or elements. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, limitation or limitations not specificallydisclosed herein.

An immunogenic composition is one which triggers either a humoral immuneresponse or a cellular (T cell) response, or both, in a human or animalto which the compositions has been administered. A vaccine (or vaccinecomposition) is an immunogenic composition, which after administered toa human or animal, which results in either no infection or infectionwithout less severe or no symptoms upon challenge with a virulent strainof the same microorganism as the vaccine composition contained. In thecontext of the present invention, cellular immune responses areespecially important in protecting a human or animal against infectionby virulent B. melitensis.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

In general the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art, unlessotherwise defined herein.

In the present context, functionally inactivated means that a gene doesnot produce a biologically active gene product (there is less than 10%of the normal enzymatic activity or ligand binding activity). In thepresent context biological activity does not encompass triggering animmune response in a mammalian host in which the functionallyinactivated gene product is expressed. However, it is intended thatfunctionally inactivated includes those cases in which the gene is notexpressed, for example, due to a large (or polar) insertion in apromoter region or other untranslated sequence.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (see e.g.Fingl et. al., in The Pharmacological Basis of Therapeutics, 1975, Ch. 1p. 1). It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions, or to other negative effects.Conversely, the attending physician would also know to adjust treatmentto higher levels if the clinical response were not adequate (precludingtoxicity). The magnitude of an administered dose in the management ofthe disorder of interest will vary with the severity of the condition tobe treated and to the route of administration. The severity of thecondition may, for example, be evaluated, in part, by standardprognostic evaluation methods. Further, the dose and dose frequency,also varies according to the age, body weight, and response of theindividual patient or animal. A program comparable to that discussedabove also may be used in veterinary medicine.

Use of pharmaceutically acceptable carriers to formulate the immunogeniccompositions herein disclosed for the practice of the invention intodosages suitable for administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular thoseformulated as solutions, may be administered parenterally, such as byintravenous injection. Appropriate compounds can be formulated readilyusing pharmaceutically acceptable carriers well known in the art intodosages suitable for oral administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions, including those formulated fordelayed release or only to be released when the pharmaceutical reachesthe small or large intestine.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levitating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Thecompositions and methods and accessory methods described herein arerepresentative of preferred embodiments and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art, which are encompassedwithin the spirit of the invention, are defined by the scope of theclaims.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with a protein or other cellular component of interest may bemade by methods known in the art. See, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories; Goding(1986 and subsequent editions) Monoclonal Antibodies: Principles andPractice, 2d ed., Academic Press, New York.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

All references cited herein are hereby incorporated by reference to theextent there is no inconsistency with the present disclosure. Thesereferences indicate the level of skill in the relevant arts.

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified articles which occur to the skilledartisan are intended to fall within the scope of the present invention.

EXAMPLES Example 1 Bacterial Strains, Plasmids, and Growth Conditions

Bacterial strains and plasmids used in this study are listed in Table 1.Strains GR019, GR024, and GR026 are the EZ::TN-lux transposoninsertional mutants of B. melitensis 16M containing the promotorless luxoperon. A schematic illustration of the bioluminescence transposon andthe mutagenesis and analysis strategy is shown in FIG. 15. B. melitensisRev-1 is an attenuated strain of virulent B. melitensis 6056 (2, 13) andis used as a vaccine for brucellosis in small ruminants (4). B.melitensis 710 is a spontaneous rough mutant of Rev-1 isolated fromvaccinated sheep and is phenotypically identical to Rev-1 except for therough LPS. GR023 is a virulent bioluminescent strain of B. melitensis16M (40) used for challenge studies. All Brucella strains were grown inbrucella broth (Difco, Detroit, Mich.). Ampicillin 100 μg/ml,chloramphenicol 20 μg/ml, kanamycin 50 μg/ml, and zeocin; 50 μg/ml forE. coli and 250 μg/ml for Brucella were added to the medium asnecessary. Brucella strains were grown at 37° C. with shaking unlessotherwise stated. E. coli strain DH5α (Invitrogen) and EC100D™pir+(Epicentre, Madison, Wis.) were grown in LB broth (Difco).

Suicide vectors pGR026-90K and pGR026-91K for generating deletions inBMEI1090 and BMEI1091, respectively, were created using pZErO-1. Toconstruct pGR026-90K, approximately 1 kb DNA sequences upstream anddownstream of the deletion target was amplified by PCR (upstream:forward 5′atcaacggtaccCGTTCAGCGCGTCGAGATCG (SEQ ID NO:1) and reverse5′gctctaggatccGACTGATMTTATGCCGTGCG (SEQ ID NO:2), downstream: forward5′acagtcgaatccATMCCGMGCCTATTCCTTC (SEQ ID NO:3) and reverse5′ggtaacctqcagCGMCGTGCCCGCAT CAT (SEQ ID NO:4)) and cloned into pZErO-1to generate plasmid pGR026-90. Appropriate restriction sites wereincluded in the PCR primers to facilitate the insertion of the kanamycinresistance (kan^(r)) gene from pUC4K between the 2 fragments to generatepGR026-90K. Bases added to the 5′ end of each primer to providerestriction sites are underlined. To construct pGR026-91K, the desireddeletion target was amplified with approximately 1 kb upstream anddownstream sequences using specific primers (forward5′agatacggtaccTCTTCCATCGTTCCGGGCCT (SEQ ID NO:5) and reverse5′catgcatctaga GACGCCGTTGATGTTCCATGTA (SEQ ID NO:6)) and cloned intopZErO-1 to generate pGR026-91. Then, inverse PCR was performed onpGR026-91 using primers (5′tcttgagaattcCCCMTGCGACCGCTT (SEQ ID NO:7) and5′gattcagaattcTTlGGCGATCCGCCTGGCA (SEQ ID NO:8)) designed to amplify allbut the deletion target. The inverse PCR product was digested withrestriction enzyme and ligated to the kan^(r) gene fragment to generatethe final suicide vector pGR026-91 K.

To construct plasmids pBBVirB4, pBBGalE, and pBBI1087-90, DNA sequencesencoding the respective ORFs were amplified using primers (VirB4:forward-5′agagagGGTA CCCATGTTCATATTGCCGCTGATCG (SEQ ID NO:9) andreverse-5′agagagGGATCCTGCTGGTTACA GTCAGGGCGAAT (SEQ ID NO:10); GalE:forward 5′agagagGGTACCAAAGCCCGGTAAAACGATTGATG (SEQ ID NO:11) and reverse5′ agagagGGATCCGTTCCGGCATTTTCTGGCAAA (SEQ ID NO:12); 1087-90:forward-5′agagag ACTAGTTGTGCCGTCGTTTCCACCTG (SEQ ID NO: 13) andreverse-5′ agagagCTCGAGAGGGACGGGGA TCGGGTTAT (SEQ ID NO:14). PCRproducts were digested with restriction enzymes and ligated to pBBR-MCS4to generate the complementation plasmids.

Example 2 Mapping of the EZ::TN/lux Transposon Insertion Site

The site of transposon insertion in GR019, GR024, and GR026 mutants wasidentified by rescue cloning. Two micrograms of genomic DNA from eachstrain was digested to completion with NcoI to generate a fragment withintact transposon and flanking sequences. Digested DNA was religatedusing a FastLink DNA ligation kit (Epicentre). Ligations were dialyzedand transformed into electrocompetent EC100 Dpir+ cells (Epicentre) andplated on LB agar containing kanamycin. Two independent kan^(r) colonieswere selected, the plasmid was extracted and the site of insertion wasidentified by sequencing the plasmid DNA bi-directionally using outwardprimers (40). Sequencing was performed using dye terminators at the DNAsequencing core facility, University of Wisconsin Biotechnology Center.Sequences were compared to the 16M genome sequence to determine the siteof insertion.

For Southern hybridization, 10 μg of genomic DNA was digested with ClaIand separated in a 0.7% agarose gel by electrophoresis. The single copyinsertion of the transposon at the expected location was detected usingthe kan^(r) gene as a probe. A 700 bp internal fragment of the kan^(r)gene was amplified from pUC4K using primers, KanF2; 5′GCTCGAGGCCGCGATTAAAT (SEQ ID NO:15) and KanR2; 5′TCACCGAGGCAGTTCCATAGGA (SEQ IDNO:16), labeled with North2South Direct HRP detection and labeling kit(Pierce) and used as a probe.

Example 3 Inactivation of BMEI1090 and BMEI1091 in 16M

To generate specific deletions, suicide vectors pGR026-90K and pGR026-91K were electroporated into B. melitensis 16M. Cells were plated onbrucella agar containing kanamycin. To select for double recombinants,the kan^(r) colonies were checked for sensitivity to zeocin (zeos). Theresulting kan^(r) and zeos clones were streak purified, and one suchpurified clone was used for further study.

Example 4 Macrophage Infection

The macrophage-like RAW 264.7 cells were cultured in RPMI supplementedwith 10% heat-inactivated fetal calf serum. For macrophage growthassays, 24-well microtiter plates were seeded with 5×10⁵macrophages/well and infected with different B. melitensis strains at1:50 multiplicity of infection. Cells were incubated for 1 hr at 37° C.in 5% CO₂, extracellular bacteria were removed with 3 washes of PBSfollowed by treatment with gentamicin 25 μg/ml for 30 min. Followinggentamicin treatment, the cells were maintained with medium containing 5μg of gentamicin/ml. At specified times, cells were washed with PBSthree times, lysed with 0.1% Triton-X, and plated on brucella agar todetermine intracellular bacterial counts. All experiments were performedin duplicate.

Example 5 IRF-1^(−/−) Mice Virulence Assay

Groups of 6-9 week old IRF-1^(−/−) (n=4) were infectedintra-peritoneally (i.p.) with 1×10⁷ CFU of GR019, GR024, GR026, Rev-1and BM710 strains. Infected mice were housed in a biosafety level 3facility and monitored for survival (virulent Brucella kills these micewithin 14 days; 21). For imaging, mice were anesthetized withisoflurane, and bioluminescence was recorded after a 10 min exposureusing a CCD camera (Xenogen, Alameda, Calif.). From the surviving mice,livers and spleens were collected aseptically, homogenized in PBS andplated on brucella agar. Plates were incubated at 37° C. for 4 days, andCFU were determined. For histology, a portion of livers and spleens werecollected and fixed in 10% formalin, 5 μm sections were prepared,stained with hemotoxylin and eosin and microscopically examined.

Example 6 Vaccination and Challenge Studies

IRF-1^(−/−) mice 6-9 weeks old (n=9/group) were vaccinated with 1×10⁷CFU i.p. with B. melitensis strains GR019, GR024, GR026, or BM710 in 200μl PBS. As a control, a group of 10 mice were injected with 200 μl PBS.Similarly, C57BU6 mice (n=20/group) were vaccinated i.p. with 5×10⁷ CFUwith each of the above strains and the Rev-1. Mice were imaged dailyusing a CCD camera until challenge. After 60 days, both IRF-1^(−/−) andC57BL/6 mice were challenged with 1×10⁶ CFU of virulent bioluminescentB. melitensis GR023i.p. Following challenge, mice were imaged with 10min exposure using a CCD camera and dissemination of virulentbioluminescent GR023 in different groups was monitored.

For IRF-1^(−/−) mice, the survival was recorded in different groupsfollowing virulent challenge. At 44 days post challenge, livers andspleens from surviving mice were processed for CFU enumeration. ForC57BU6 mice, to determine CFU in livers and spleens, 4 mice from eachgroup were killed at weekly intervals. Portions of the livers andspleens were weighed and then homogenized in PBS. Homogenates wereserially diluted, plated on brucella agar with or without antibiotic andcolonies were counted after 72 hr of incubation at 37° C. To determinethe histological changes at each time, a portion of livers and spleenswere collected, fixed in 10% formalin, and 5 μm sections were preparedand stained with hematoxylin and eosin.

Example 7 Identification of Brucella-Specific MHC Class I-Restricted TCell Epitopes

Raw264.7 mouse macrophage cells (haplotype H2^(d)) are infected (MOI1000) with B. melitensis cells for 48 hrs. Infected cells, along withuninfected control cells (2×10⁹ each) are harvested by scraping, andmembrane proteins are extracted using Mem-PER (Pierce Chemical Co.,Rockford, Ill.). The extract is dialyzed overnight in 0/5% CHAPS bufferto prepare for immunoprecipitation. H2-D^(d)/peptideco-immunoprecipitation is performed using Seize PrimaryImmunoprecipitation Kit (Pierce) couple with anti-mouse H-2D^(d)monoclonal antibody that recognizes a conformationally sensitive epitopeof H-2D^(d) (5589125, BD Biosciences Pharmingen, San Diego, Calif.).After elution of the MHC I/peptide complex in acidic conditions, thepeptides are separated from MHC I components by passing through a 5 kDaMWCO filter (Millipore, Billerica, Mass.). Micro BCA protein assays areperformed on the peptide mix, and the peptides are separated, sequencedand analyzed by liquid chromatograph/mass spectrometry.

Alternatively, the RAW264.7 mouse macrophage cells are infected withinvasive E. coli expressing GFPuv for identification of infected cells(MOI 100 24 hr infection). The results demonstrated that MHC I andassociated peptide can be identified, and the invasive E. coli vaccinevector can be used to deliver antigen to cells for processing andpresentation by MHC class I. In this example, of eight H2-D^(d) nonamersfrom Infected Raw264.7 cells, one (NYNSHNVYI, SEQ ID NO:17) was specificto the GFPuv protein. Other sequenced peptides included HYLSTQSAL (SEQID NO:18), LFTGWPIL (SEQ ID NO:19), KFICTTGKL (SEQ ID NO:20), DFKEDGNIL(SEQ ID NO:21), LPVPWPTLV (SEQ ID NO:22), EYNYNSHNV (SEQ ID NO:23) andTPIGDGPVL (SEQ ID NO:24). TABLE 1 Bacterial strains and plasmids used inthis study. Strains or Plasmids Descriptions Reference or source Strains16M Wild type strain of B. melitensis ATCC DH5α E. coli strain used forcloning Invitrogen EC100Dpir+ E. coli strain used for rescue cloningEpicenter GR019 Bioluminescent B. melitensis with EZ::TN This studytransposon inserted in the virB4 gene GR023 Bioluminescent B. melitensisstrain used for 40 mice challenge studies GR024 Bioluminescent B.melitensis with EZ::TN This study transposon inserted in the galEhomolog GR026 Bioluminescent B. melitensis with EZ::TN This studytransposon inserted in the intergenic region of BMEI1090-1091 BM710Spontaneous rough mutant of B. melitensis This study Rev-1 strain Rev-1B. melitensis 16M vaccine strain  2 GR-1090Δ B. melitensis 16M withBMEI1090 replaced This study with Kan^(r) GR-1091Δ B. melitensis 16Mwith BMEI1091 replaced This study with Kan^(r) Plasmids pBBR-MCS4 Broadhost range plasmid. Ap 25 pZErO-1 Cloning vector. Zeo Invitrogen pUC4KSource of kanamycin resistance marker. Km Amersham pGR026-90 pZero-1containing BMEI1090 with This study approximately 1kb upstream anddownstream sequences. Zeo pGR026-90K pGR026-90 containing kan^(r) markerreplacing This study ORF I1090. Zeo, Km pGR026-91 pZero-1 containingBMEI1091 with This study approximately 1kb upstream and downstreamsequences. Zeo pGR026-91K pGR026-91 containing kan^(r) marker replacingThis study ORF I1091. Zeo, Km pBBVirB pBBR-MCS1 containing the virBregion used 34 for complementation. Cm pBBVirB4 pBBR-MCS4 containing B.melitensis virB4 This study ORF used for complementation. Ap pBBGalEpBBR-MCS4 containing B. melitensis galE This study ORF used forcomplementation. Ap pBBI1087-90 pBBR-MCS4 containing B. melitensis Thisstudy BMEI1087-90 ORFs used for complementation. ApAp; Ampicillin,Zeo; Zeocin,Km, Kanamycin,Cm; Chloramphenicol,

TABLE 2 IRF-1^(−/−) mouse virulence assay. # of mice # of mice Tissuedamage^(a,b) CFU counts^(a) Strains infected survived Liver Spleen LiverSpleen 16M 4 0 Severe Severe 1.9E+10 1.6E+10 GR023 4 0 Severe Severe1.5E+10 1.4E+10 GR019 4 4 None None 1.3E+03 1.0E+04 GR024 4 4 None None9.5E+03 1.2E+04 GR026 4 4 Minimal Minimal 1.3E+04 3.2E+05 BM710 4 4 NoneNone 2.0E+02 4.5E+03 Rev-1 4 0 ND^(d) ND ND ND GR-1090Δ 4 2^(c) (1090)ND ND 6.6E+04 1.1E+06 GR-1091Δ 4 0 (1091) ND ND ND ND^(a)CFU counts and tissue damage were assessed two weeks post infectionexcept for the 16M and GR023 infected groups for which tissues werecollected when mice were moribund.^(b)Liver damage was assessed based on the number of focal granulomasand spleen damage was assessed based on the loss of architecture ofwhite and red pulp. Tissue sections were visualized at 4X magnificationand damage was assessed by observing more than 5 fields of view.^(c)Survived for at least 3 weeks.^(d)Not determined

TABLE 3 Liver and spleen damage in C57BL/6 mice vaccinated withdifferent Brucella strains following a virulent challenge. Weeks PCTreatment Liver^(a) Spleen^(b) groups 1 2 3 4 1 2 3  4 GR019 ++ +++ +++++++ +++ ++ ++ + GR024 ++ ++ ++ + ++ ++ ++ + GR026 ++ ++ ++ + +++ ++ + −Rev 1 + ++ ++ + ++ +++ ++ + 710 +++ ++++ ++ ++ + ++ ++ ++ Control ++++++++++ +++ ++ ++ ++++ ++++ ++^(a)Livers were scored by the number of focal granulomas observed perfield of view at 4X magnification. At each time, 8 fields were countedto determine the number of granulomas: (+) 1-8; (++) 9-16; (+++) 17-24;(++++) 25-32; (+++++) 33-40 granulomas.^(b)Spleens were scored on loss of white and red pulp architecture. Ateach time, 8 fields (4X) were scored using the following criteria: (−)normal spleen or no noticeable changes; (+) enlarged follicles,increased cellularity, and white pulp; (++) hyperplasia, with asignificant increase in follicle size, and white pulp; (+++) increasedred pulp, early loss of architecture, and the diminution of white pulp;# (++++) severe loss of architecture, and a dramatic reduction in thenumber of follicles.

TABLE 4 Brucella peptides identified from MHC I molecules* BMEII0160Flagellar Hook-associated protein BMEII0148 Extracellular serineprotease BMEII0793 Multidrug resistance efflux pump BMEI1895 Outermembrane protein BMEII0976 ABC transporter ATP-binding protein BMEI1715Maltose transport permease BMEII0862 Dihydrodipicolinate synthaseBMEII0126 Amino acid permease BMEII0348 4-aminobutyrate aminotransferaseBMEI1744 Glucose-resistance amylase regulator BMEI1427UDP-4-dehydro-6-deoxy-2-actamido-D-glucose BMEI0943 VitaminB12-dependent ribonucleotide reductase BMEI0864 NifR3 Nitrogenregulation BMEII0749 Hypothetical protein BMEI0903 Hypothetical proteinBMEI1718 Hypothetical protein*Examples from over 2,500 different peptides isolated from MHC IH-2K^(d)

TABLE 5 Nucleotide sequence encompassing two open reading frames of theBrucella melitensis 16M genome (SEQ ID NO:26). LOCUS AE009548 10975 bpDNA linear BCT 20-MAR-2003 DEFINITION Brucella melitensis 16M chromosomeI, section 105 of 195 of the complete sequence. ACCESSION AE009548AE008917 VERSION AE009548.l GI:17983048 ORGANISM Brucella melitensis 16MAUTHORS DelVecchio et al. 2002. The genome sequence of the facultativeintracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA99:443-448) FEATURES Location/Qualifiers gene complement(6688. . .7740)/gene = “BMEI1O87” CDS complement(6688. . .7740) /gene = “BMEI1087”/EC_number = “3.2.1.52” /Product = “BETA-HEXOSAMINIDASE A” /protein_id= “AAL52268. 1” /db_xref = “GI:17983056” /translation= “MIQQDNSRKSRMKECKAWIAGISGTKLTPDEIAFFRDETPWGFILFARNVESLEQVSELTAHLRDLTGLDQTPVFIDQEGGRVQRLRPPLVPNYPSASEIGAIYARDKEKGLRAAWLHARLHAFDLLKVGVNADCLPVLDVPVEGAHDVIGMRAYSKNPHAVAEMGRAAAEGLLAGGVLPVVKHMPGHGRAFSDTHKELARVSVALNELVAHDFVPFKALNDLPMAMTAHVVFDCIDPERPSTLSPTVINTIIRDVIGFDGLVISDDISMKALSGDLGDITDGIVTAGCDIVLYCSGVMEELVAARVPVLDGKAKRRAELAEVYAGDPDLSDEDEVRAEFNAMFEPIA” gene complement(7908. . .10817) /gene = “BMEI1O88” CDScomplement(7908. . .10817) /EC_number = “3.2.l.” /product = SOLUBLELYTIC MUREIN TRANSGLYCOSYLASE” /protein id = “AAL52269.1” /db_xref= “GI:17983057” ORIGIN     1 aggctgccat tgctcaaaat caatgcaact gaagccgttccgacaaaagc gcgaagcggt    61 tttttggaat catcctcaaa caaaatcttg gagcgggatgatggttggac ttaaattcaa   121 cccgttttag agcgcgtttc gatctgattg aatcagatcggcgctctaat cctttgtttt   181 gacgcgcatc ttttccgaaa accgtttcac acttttcgggatgcgctcta aagaacggaa   241 gacgtgcctt cgatgaacgg ctgatatcga accggcatgaggtcttcctg ttcaaaacgg   301 cttccaacct ttgaaatccg cgtcatgatc tggcgtccatcgccagggcc gatcggggct   361 atcaggacgc catgggtggc gagcagttca acgaaatggcgcggcacctc atcgcatgcg   421 agccagatga caatgcggtc aaacggcccg cccggcataccgtggcgccc gtctgtatgt   481 ttcaccatga tattctcgcg cttcagcgaa acgaactgctggagagcgtg gtcgcagagt   541 tttcgatacc gttccaccgt cgttacacgg ccggacagcaaggacataac ggcggcggta   601 aagccggagc cggtgccgat ttccagaacc cgatggccgggctcaagctt cagggcggaa   661 atgacgcgcg cctgatcgtc tatgccttcc atatattcaccgcaatcaag cggcgcggtt   721 cgcgggctat aggcaagatg cgaccatgcc gccgccagaaagctctggcg cggcgttgct   781 tcaattgccg caaaaagttg cggatcatca atgctgtgcccacgcatccg cagaacaaag   841 gatgcaaatc cctcccggtc cgaaagccgc gggcgttcagacgttgcctg cctcatgctt   901 ccactccaag cgccgcgccc agttctgcac gaaccttatgagcggtcaga tcaaggtgga   961 gtggggtcac tgaaatgcaa cccgaacgga tggcagcaatatcgctgtcg tcggcaaccg  1021 gagccttgcc gcgaccgaaa tgcagccaga aataagggaaaccacgtcca tcgcggcgct  1081 cgtcaaggcg cgcatcatgg ctaagcttgc cttgtgccgtgacgcgcacg cccttcactt  1141 cttccggagc gcaattcggg aaattgaggt tcaacagcacgccttccggc cagcccgcct  1201 ccatcagcct cccgataagc tcaggcgcat gagcttccgccgtttcccac ggcacgatcc  1261 ggcgatcgcc cgcatattca tattcctgcg acaaagcgatggctcgcaca ccaagcaatg  1321 tcccctccat cgcaccggca accgtgcccg aataggtcacatcgtcggcc atgttcgccc  1381 cggaattgac gccggagagg acgagatcgg gcgcgcccggcaatacatgg cgcaccccca  1441 tgatgacgca atcggtcgga gtgccgcgca gggcaaaatgacgggcatcg atctggcgaa  1501 ggcgaagcgg ctccgacagt gtcagtgagt gggcaagcccgctctggtcc gtttcagggg  1561 ccaccaccca cacatcgtcg gagagcttgc gtgcaattcgctccagaaca gcgaggcctt  1621 cagcgtggat accgtcatcg ttcgtcagca gaatacgcaatttgtcactc cttcgccgaa  1681 atggataaga cacttaagac actacagcgg ttccagttgaaatgggatcg ttgaaactgc  1741 tctctctttg ttctttcgca tgtccccaaa accggttcccacttttgggg gcatgctata  1801 attccagatc aagcggcttt ttcgatccgc gtgaggccgcccatatatgg ctgtaatgct  1861 tcaggaatat gaatgctgcc gtcttcctgc tggtaattttccataaccgc aatcagcgcg  1921 cgcccgacag cagcgcccga cccgttgagg gtgtgcacgaagcgcgtgga tttttcgcct  1981 tccgggcgat agcgggcatt catgcggcgg ccctggaaatcaccgcaggt cgaacagctt  2041 gaaatttcgc gataggtgtt ctgccccggc aaccagacctcgatatcata ggtccgctgt  2101 gcgccaaagc ccatgtcgcc cgtgcaaagc acaacggtacggaacggcag gcccagccgc  2161 ttcagcactt cttccgcgca agccgtcatg cgctcatgctcggcaacgga gctttccgca  2221 tcggtgatcg ataccatctc cactttcagg aactgatgctggcgcaacat gccgcgcgta  2281 tcgcgcccgg ccgaccccgc ttccgagcga aaacatggggtcagcgccgt gaagcgcagc  2341 ggcagcccct tcatatcgac aatttcttcg gcaaccagattggtgagcgg cacctccgcc  2401 gtcgggatca gccagcggcc atccgtcgtg cggaaaagatcttctgaaaa cttcggcaat  2461 tgccccgtgc catagaccgc ttcgtcgcgc accatcagcggcggcatgac ttcggtataa  2521 ccgtgttctg tcgtgtgaag atcgagcatg aactggccaagcgcgcgctc aagacgggcg  2581 agcgggcctt tcagcaccgt aaagcgcgca ccggcaagcttggccgcgcg ctcgaaatcc  2641 atgtatccaa gcgcctcgcc aagctcaaaa tgctctttcggctggaagga gaaattgtgc  2701 gggttgccaa tgcggcgcag ctcaacattg tcgctttcatccttgccgag cggcacatca  2761 tcaagcggaa tattgggaat ggtggacaat gcgtcgctcagttccttgct gaggcggcgc  2821 tcgtcttctt ccgcatgggc gagaaaatct ttcagttcgcccacttcggc cttcagcttt  2881 tcagccgtgc ccatgtcctt tgcggccatg gccttgccgatttccttcga ggcggcattg  2941 cggcgctcct gcgctgcctg caccttgccg acatgctcgcggcgcttttc atccagcgca  3001 atcagttcgg acgaaagcgg agcagcccca cgctttgcgagcgccttgtc gagggtttcc  3061 gggttttcgc gaatccattt gatgtcgagc atggaaaaaagccatttcgt gaaattgaac  3121 agaagcgagg ctaaacgatc ttcagcccca aagatgcctgacgtcagatc aggtggagga  3181 agcgttgtta tcagcgtcgg cagatgcctg cgcctcatcccgcttcttct cgatcatgcg  3241 cgccagaaag atcgaaatct cgtaaagaag gatcgtcggcaaggcaagac cgatctggct  3301 cgccgggtcc ggcggggtca gcaccgcagc cgcgacgaaggcaatgacga tcgcatattt  3361 gcgcttgtcc ttcagccccg ccgaagtcac cagccccacacgcgccatga ggctcgtcac  3421 caccggcaac tggaagacca ggccaaaagc aaagatgagcgtcatgatga ggctcagata  3481 ttccgacact ttcggcagaa gcgaaatctg gacctcgccgctgccgccgg tctgctgcat  3541 ggcgaggaag aaccacatca ccatgggcgt gaaaaagaaatagacgagcg cgccgccgat  3601 caggaacaga atgggcgacg cgatcaggaa cggcagaaatgcagtgcgtt cgtgcttgta  3661 gagaccggga gccacgaatt tataaatctg tgcggcgatgaccgggaggg ccagcacaat  3721 gccgccgaac atggccacct tcacctgcgt gaagaagaattcctgaggtg cggtatagat  3781 caattccgcc ttggagcggt ccatgccggc ccagtcgatggcccattgat acggcaccac  3841 aagcaggttg aagagctgtt ttgcgaaagc aaagcagaaaatgaatgcca cgaaaaaagc  3901 caggatagcc caaataaggc ggcggcgcag ttcgatcaggtgttcaagca gaggcgctgc  3961 gctctgttcg atttcatcct cgtcccggtt cacgctttggttcctgtctt ctttgtggtc  4021 tttttaaccg gcgttgcggt cttgtctgcc gtcggcttgggggtagctcc ggtttttttg  4081 gcagtctttg tcgtcgtcgg tttcggcccg gcttttgcagccggacgcgg tgatgttttc  4141 ctaggcttgg cgggttcttc gggcgcggtg atcattggtacgggaactgg cggcgcggga  4201 actggcgttc cgcccggctc aaccggcgtc gtaacctcacccaccttgtt ctcggtgact  4261 ggcgacattg atgttgcgga ctggagacca gaccgcaaatcctcgccagc actgcgaatc  4321 gggtcaaaaa cctgtgtcag ccttgtgcgc ggatcaaggcttctggcttc atcgatgatg  4381 gtcttgacgt cttcaagttc cgcctctttc aaggcctcgttgaattgatg gcgaaactcg  4441 ttggcggtgg tgcgcatgcg tgcagtcgcc ttgccgaacgcgcgaagcat tttcggcaaa  4501 tccttgggac cgaccaccac aatcatgaca attgcgataatcagcagttc agaccaagcg  4561 atatcgaaca taatttgata ccttgcgctc tgcgcgcacatcctgtctct tggcgaaaag  4621 ccgcactgcc cacaaacctg ccatgcgcgt tttcagcccatggcagttca tcccggaagg  4681 atcaggactt ggtggtcttc ttgacgtcct tgacgggttcttccgctttg gcgtcgatcg  4741 tacgcggatc ttccttggcg tcttcgtcag ccatgccctgcttaaaattc ttgataccct  4801 tggcgacatc gcccatcagc tcggggatct tgccgcggccgaacagaaga agcacaaccg  4861 ccagaacgat cagccagtgc cagatggaaa agctacccatattattcctc tcagtgccgc  4921 ccaaggcgcg gcatatgcct gctatctccg atacgatttaagcgctttca acaaatcttt  4981 caaacagaag tgtgatgatg aacggcttca aaccggattaattcgtcgca ggcagaaatt  5041 ttgttctatt ctcccctggg tgcaagcaaa cccagtccctccagatcaat atcctccagc  5101 gggtcctccc cttcggtcag ctcgtccggg tcgatattggggatcggtac ggcaaaactg  5161 gaaggaatgc gcgccgagag aagccctgcg ccgcgcaattcctcaagacc gggcagatcg  5221 cggatttccg gcaggccaaa atggtcgagg aaagcgtcggtggtgccata tgttaccggg  5281 cgccctggcg tgcgcctgcg cccgcgcagc ttgatccagccggtttccat caagacatca  5341 agcgtcccct tggatgtttc cacgccgcga atatcctcaagttcggcgcg tgtcaccggc  5401 tggtgatagg caatgatggc aagcacctcc atggtcgcgcgcgaaagctt gcgctgctga  5461 acagtctcgc ggttcatgat gaaggcgaga tctggcgcggtgcgaaacgc ccagccactg  5521 cccaccttca caaaatgcac gcccctgccc tcgtaaaccttctggagatg gttcaaaacc  5581 ggagcaatat ccacattggc gggaagccgc tcggcaagtgcgcgctcgca aacaggctgc  5641 gaagacgcaa aaacaatcgc ctccacaatg cgggcaagctcggcaagcgt caccggcgag  5701 gcaggccccg cctgctcttc ttccccaacg ccttccatatccatcaaatc gcggcgctct  5761 gcttcaggca ttttcgtcct catcgaattc atcgagttcgcgggtcgcgc gcatatagat  5821 cggctcgaac ggagcgttct ggcgtacttc aagcttgccttcgcgcacca gctcgaggca  5881 tgcggcgaaa gaactggcaa gcgccgacgc cctctcctgcggagaaagtg cataatcgat  5941 caaaaaacgg tccagcgaaa cccagtcgcc caccgcgcccatcaggcgca caagcgccgt  6001 gcgtgcctcc ttgagggacc agacgctgcg tttttctatctgtacctggg aaaccgcctg  6061 gcgctggcgc tgcgacgcat aagcgctaag cagatcgtaaagcgttgcgg aaaaacggct  6121 ggcgcggtcc accaccacca tttccggcat gccgcgcgggaaaacatcgc ggccgagccg  6181 atgacgattg acgagtgccg ccgccgcatc gcgcatggcttcaagccgtt tcaaccggaa  6241 ttgcagggag gcaacgagtt cctcgcccgt ggcgccatcgtcgccctgct gcttcgggat  6301 cagcagcttg gatttcagat aggcaagcca tgccgccataacgagataat cggcggcaag  6361 ctccagacgc agcgcgcgcg cctgctccac gaaaccgagatattgttcgg caagcgccag  6421 cacggaaatg cgcgcaagat cgacgcgctg gttacgcgcaagatgcagaa gaaggtcgag  6481 cggaccttca aagccctgca catcgatcag cagtgacggctcgcctgcgc ctcgcccggc  6541 ctcattttgc cacagggtat ccatcggcac gcgtgtgccgtcgtttccac ctgtatgtgc  6601 atccgatgct gccaagcctg tcgtcctgct gttgcccctgccggcacaga ttctgccagc  6661 aggaacaagt ttatcaagtt ttgccgatta cgctatcggttcaaacatgg cattgaattc  6721 ggcgcgcacc tcgtcctcgt cggaaagatc ggggtcgcccgcatagactt ccgccagttc  6781 ggcccggcgc tttgctttgc catccagaac cggcacacgggcggcaacct ttaccagttc  6841 ctccataacg cctgagcaat agagcacgat atcgcagcccgccgtaacga tgccgtcggt  6901 tatatcgcca agatcgccgg acaatgcctt catggaaatgtcgtcactga tgacaaggcc  6961 gtcgaacccg atcacatcgc gaatgatcgt attaataaccgtcggcgaaa gcgtggacgg  7021 tctttccggg tcgatgcaat cgaacaccac atgggcggtcatggccatcg gcagatcatt  7081 gagcgccttg aacggcacga aatcatgcgc aaccagttcgttgagcgcaa cgctgacccg  7141 cgccagttcc ttatgcgtat cggaaaaggc gcggccatggcccggcatat gcttcacgac  7201 gggaagaacg ccgccagcca gaagaccttc ggcggcagcgcgtcccattt ccgcaaccgc  7261 atgggggttt ttggaatagg cccgcattcc gatcacatcatgcgcgccct ccaccggcac  7321 atccagaacc ggcaggcaat ccgcattgac gccgaccttcaacagatcga aagcatggag  7381 ccgggcatgg agccaggcgg cacgcaaccc cttttccttgtcgcgtgcat agatcgcgcc  7441 aatttcggac gcggacggat agttcggtac cagcggcgggcgaaggcgct gcacgcgccc  7501 gccctcctga tcgatgaaaa ccggcgtctg gtccagccccgtcaggtcac gcagatgggc  7561 ggtgagctcg ctcacctgtt cgaggctttc cacattgcgagcaaaaagaa tgaagcccca  7621 cggggtttca tcccggaaga aggcaatctc gtccggggtgagcttcgtgc cggatatacc  7681 ggcaatccat gccttgcact ctttcatgcg gcttttcctcgaattgtctt gctgaatcaa  7741 actcgcctcc gccgggttta gccgaaaaaa cggccgcttggtaggctgta ggctgtggtg  7801 acgaattaac ttatggttcg gtatgaacga aaatgctcaatagcccggca gatgcgaaaa  7861 gggcggctga cgccgccctc aaatggctgg aaccttgctggttgttttta ctgcgtcacg  7921 aaacagcttc cgccagccga cttgagacgg ctgcaaagcgccaatgcatc ttccttcgag  7981 ccagcctgta cgcgaacacg gtaataggtg cccttgccctgaatgtcggc gcgcttgata  8041 tcgacgctat gaccgccaat cacactggca tatttctgggctatgttggc ataggacttc  8101 tgcgccagct cagcagaagg ctgcgaggca atctggatgaaataaccacc cgccccggct  8161 gccgatgcga cctgcggtgc agccgatgcc tgcgcgcgctgcggcacgtt accgacgata  8221 ttgacgggct gttcagcggg gcgcgacggc acgatcggagcgcgggtcgg aaggcgtggg  8281 gtctccggcg tcgcggattg ctgtgcctgt ggcgctggcggttcatttcc tgccgcgagt  8341 gcgccgattt catcccgtgc tgccggtgcg gcaggcggcgccatattgtc ggctaccgac  8401 ggctgtgctg catgtccgaa cgaaggctga atgatcgtgccatccgggcg aacgatcatc  8461 gtttcgacct cacgcggctg gatcagcggt tcatgcgtgccggaatgagc ctgttgcgca  8521 tcgttctgcg gtacattgcc acccggttct tctgtggcattatattcgct gtcatcggta  8581 ccggaaatat cgaccggttc ttcaccggat gtaatcagggctttctgttc cgggttgttc  8641 ggaagcgttc cggccacacg gtcatagacc gccttatcctggttcggaac cgtggttccg  8701 cccggatttt ccggctgcat cttgatgggc tggttatcggcgcgaatcac aaccggctca  8761 ccggagcctc cgccgccgag gaaatgatag ccgattccgccgagcagaac cgccacaccg  8821 gcaacacttg ccaaaatcaa gccacggcga ccacgaaccgggcgattgcg gtaagcttcc  8881 gccgcgccgc ccagatcgtc ttccgtcggc attgcggcgcgctcgccata atcgccgcct  8941 tccatggtct gcgcaccctg cgcggcccag tgattgtagaaatcatcctg gctggcagtc  9001 gttgcagcgg caggtgcagc ttcagacccg gcgcgtcggtaggaagcagc agccgctgcc  9061 gccgcagcac ccaagccggc cgcggccatg ccgctattcggcatataggt cgatgcgctt  9121 tcacggaaga tgtcctcaaa agccctgtcg gcttcgctctggccttccgt gatctgcaca  9181 ttctcatcaa caccaatcgt gctgaaaact tccgcgaactccgcttccag ctcactgaga  9241 ttggtgcccg cttcctcttc gccgtaattc acttccggcagatcgagcga atgcgtctgt  9301 tcgaccttgt tttccgtgac cgtgagggtc tcaacctccggtgcaggctc cggtgcgggc  9361 atacgcgccg gagcctgcat gtcggtataa gcggcaaaggagacagcagg gcgataatcc  9421 tcgccggaat ggattgcgtg cgcataggct ggcgcgggagaagcttcttg ctcctcctca  9481 tggagatcaa gttcaacatc ggtgaagaaa tcttcatcgttgaaaaaatc ttcatccgca  9541 gtatcggcga cgtctgcgtc gaatgtgtct ccagacggctcgaaaccgaa atcatcttcg  9601 gtcaggctga tttcgtcgag ccccgacagg tcatcatccgtcgattcggc ggcggcagcc  9661 gtttccggct ctgcttcaaa ggtaaaatcg tcctcaagcgaaaactcatc ctcgattggc  9721 cggaacggat cctgggcaac aaaatcctgc acaataggcgcctggggcgt tttcagttgc  9781 ggccccgagg agagcacacc gggggcggct acacccggcgcaaaattgct gcgcggataa  9841 tagggatagg ccggcgcctc gccagtgcgt gacgcatagccctgcccggc atgggacgga  9901 gcatccagat gcggttcgct gaccggcgct tccggttcgccctgatgcgg ttgttccggc  9961 gcgtaggaaa caggctcgac gtgatccgaa tagctgttccgggatgccac aggctgcggc 10021 tcatcgccaa acagaaggtt ttccagctcg tcttcaagcgaaagcggttg ctgtgcaggc 10081 tcagccgcga aatgcgtagc gggagcatct acaggctgctgcgcattcca attattctgt 10141 gctggccagt cattctgttc cagcctgtca ttctgtgcaggccactcatc ctgtgcctgc 10201 caattgtcct gcacgccggt atcttcacgc caagcttgctcacccggctc aatggcaggc 10261 tgatagacgg tcggatcata ttcgcccgca tctgcctctatcggctgctg gcgatcacca 10321 taagccgcag gtgatgccat atgggcgtgt tcgctcgaatcacgagcctg cgaataatcg 10381 ttatagtcga attgcggaac gggctcggag cgatccacgtcctcgaactc gaaaatttcc 10441 ggtgacggtg ctgcttctgc cgagccaagg tcgagatcgaattcttcttc cagcgcggca 10501 gcgaacgcat cctcctccaa cggagactgc tcgccataggcccgctcccc gtgcacgcca 10561 aaggttgcgg tcgaaacggt ttcgctatga gctgtaggctgcgtataatc gtcgaaatgc 10621 cccataagct cgcgctcaag atcgagaacg ggatcaaaagaggggtcatc ctgcgccgaa 10681 tcaaagcgcg gctctgctcg gccctgatcc tcgaattgactgtcatggcg gcgctcattc 10741 cgagcgacgt tatcatcagc aggcgtgtcg aagtccataatccgcgaaag ttccatcagc 10801 ggatcatctt cgtgcaccgg acgctcgccg taattacggggatttgcact gctgtccgtc 10861 atggcgtgtt cctaactcaa accctggacg ccgcaagacgtctccataca ttgcatatta 10921 gcgaggcaat gtgggcaaaa gttgacggaa gtttcctgcacaggaaggaa gatcc

TABLE 6 Nucleotide sequence encompassing four open reading frames fromthe Brucella melitensis 16M genome (SEQ ID NO:27) LOCUS AE009549 10209bp DNA linear BCT 20-MAR-2003 DEFINITION Brucella melitensis 16Mchromosome I, portion of section 106 of 195 of the complete sequence.ACCESSION AE009549 AE008917 VERSION AE009549.1 GI:17983058 SOURCEBrucella melitensis 16M REFERENCE 1 (bases 1 to 10209) AUTHORSDelVecchio et al. 2002. The genome sequence of the facultativeintracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA99:443-448 (2002) FEATURES Location/Qualifiers source 1. . .4500(excerpted herein) /organism = “Brucella melitensis 16M” /mol_type= “genomic DNA” /strain = “16M” CDS complement(159. . .1916) /gene= “BMEI1089” /EC_number = “6.1.1.19” /product = “ARGINYL-TRNASYNTHETASE” CDS complement(2138. . .3346) /gene = “BMEI1090” /EC_number= “3.1.5.1” /codon_start = 1 /product = “DEOXYGUANOSINETRIPHOSPHATETRIPHOSPHOHYDROLASE” /protein_id = “AAL52271.1” /db_xref = “GI:17983060”CDS 3513. . .3839 /gene = “BMEI1091” /product = “HESB PROTEIN” CDScomplement(3920. . .4411) /gene = “BMEI1092” /product = “hypotheticalprotein” /protein_id = “AAL52273.1” ORIGIN    1 tattagcgag gcaatgtgggcaaaagttga cggaagtttc ctgcacagga aggaagatcc   61 atgactttca atcatactgatcttccttca cattagtctt actgtcactc atccaaaggc  121 gtctttcgac tttcagtacttcggacgacc gtgtaatcct agcgcatctc cgtaggagca  181 tccgcgccga taatcgtcaatcctgacgtc agcacatcgg aaacaacctg caccagccct  241 agcctggcca gcgacaagtctggatcgtta accttaataa aacgtaagtc cggattttcc  301 gcgcctctgt tccattgcgaatggaacgaa ctggcgaggt cgtagaggta gaaagccagg  361 cgatgcggct cctgatgaatggctgccgat tcgatcaggc gcgggtattc cgcaagcttg  421 cgaacgagcg caatttcgctctcgtcggtc agcttttcaa aatgcgaccc catggccacg  481 cggtcaagat cgacaagcccaagctggtcc gcagcctgac ggaaaaccga atggcagcgc  541 gcggaagcat attgcacatagaaaaccgga ttgtccttgg actgctccgt caccttggcg  601 aagtcgaagt ccaatggcgcatcgttcttc cggtaaagca tcatgaagcg gaccggatcg  661 cgaccgacct cgtccaccacatcgcgcagc gtaatgaact cgcctgcccg cttggacatg  721 cgcaccggct cgccattgcggaacagcttc acgagctggc acaggagcac ggtcaatttg  781 gccttgccat cggaaacggcacgcgcaacg gcttccagac gcttgacata accgccatga  841 tccgcgccga gcacatagatcatctcattg aagccgtggt cgtacttgtc cttgaaatag  901 gccacgtcac ccgcaaaataggtgaacgag ccatcggact tcatcagcgg acggtcaata  961 tcatcgccca cttccgtagaacggaacagc gtctgctcac ggtcttccca atcttccggc 1021 aactgcccct tcggaggcggcagcttgccc ttataaacat ggcccttgag cgtcagatca 1081 ttgatcgcgt tacggatcgcgcgcgcatgg tcgacatgta gcttgcgctc ggaatagaag 1141 acatcatgat gcacgttcagcgcgtcgaga tcagcgcgga tcattgccat catggcgtcg 1201 atcgtgcggt ccttcacgatggccagtgct tcggcttcag gcatttccag aagttttgtg 1261 ccaaactcac cggcaagctcctgcccgacc cgcacgagat aatcaccggg gtaaagcccc 1321 gccggaatct cgccgatgctttcgcccagt gcctcacgat agcgcagcat cacagaacgc 1381 gcgagaacat cgatctgcgcgcccgcatcg ttgatgtaat attccttgac gacgtcatag 1441 cccgcgaatt tcagcaggttcgccagcaca tcacccacaa ccgcgccccg gcaatggccg 1501 acatgcatcg ggcccgtagggttggccgat acatattcga cattgacctt cttgcccgcg 1561 ccaagcctgg agcggccaaaatccgttccc tcgttcagca tcaccaaaag ctcgcgctgc 1621 caatagctgg ccttgaggcgcagattgatg aagcccggac cggcgacatc gacggattcg 1681 acatcctcat cggccttcagcgcctcggca atgcgggcag caagctcgcg cgggttctgg 1741 ccgaccgcct tggaaagcaccattgcggca ttggtcgcga tatcgccatg cgaagcatcg 1801 cgcgggggct cgacacctatgcgtgaaagg tcaagttcac caccatcttt tggtttcaga 1861 tcaatatctt gcaacgtttttttaatacgt gcatcgaaat ctgcaaagat attcatggtc 1921 tgtcctgtca ggctagcgcggttcctgttt taacagaatc gccggaacca ctctaactat 1981 tgttttgtcg cattttccaacgcaaaaccg tttcacactt ttggctcgaa aatactctaa 2041 cgcctggatt tttttccagttttcccggcg cgggttcatc caaaaaacgc ggatattcga 2101 tgcatttgcg tatcgaagccgcttcgtccg gccccgatta agctaaatcg ggggttcggt 2161 caaacaatcg tcggtgttcgcgcacggcat aattatcagt catcccggcc agataatcgg 2221 ctacgcggcg tgcgagtgctgccttatcca gtgcctcaca gcccaaacgc cattcatcag 2281 gcatcaatga gggatcggtgaaacaggcat cgaagagatc ctgcacgatc ctgtcggctg 2341 cgtgcctgcg caccaccacgctttcgtgaa aatagagatt cttgaacaaa aagcgcttca 2401 gcaccttttc ctcggcccgcatggcgtcgg aaaagccaac cagcgcgcgc ggctggttgt 2461 gcacgtcttc catcgttccgggcctggcgg atgcaaggcg gcgctgcgcc tcctcgatca 2521 cgtcttccac catgatcgtgatctggcggc gcaccagttc gtgtccagtg cggacggggt 2581 cgagattggg ataacgtgtccgcacaatat caagcagccg tttggcgagc ggtacttcgt 2641 ccagcgattc gagggtcaagagccctgccc gcaagccatc atcaatgtca tgcgcattgt 2701 aggcaatgtc gtcggcaatggccgcgcatt gcgcctcaag gctcgcaaag cgtgaaagct 2761 ccagatcata gcgcgcgttaaaatccagaa tgggttgcgg aaccgggata tcgggatggg 2821 ctgcatatgg ccccagcaacgggccattat gcttcaccag accttccagc gtttcccacg 2881 aaaggttgag gccatcgaaatcagcgtagc gatgctcaag cttcgtgacg atcctgagcg 2941 actgggcatt atggtcgaaaccgccgaaat tcttcatgcg ctcgttgagt gcgtcctcgc 3001 cggtatggcc gaagggcgtgtggccgaaat catgaacgag agcgacagct tcagcgaggt 3061 cttcatccag gcgcagcgcgcgcgccagcg cccgcgcaat ctgcgccacc tcgatggtgt 3121 gcgtcagcct cgtgcggtaatgatcgccct catgcgcgat gaaaacctgc gtcttgtgct 3181 ttaaacgccg gaaagccgtggagtggataa tacggtcacg gtcccgctgg aacggcgtgc 3241 gggtcgggct ttccggttccggcaccagcc ggccacggct gaaagcagga ttgctggcat 3301 aaggcgcacg ttcgcgataaccgaagccta ttccttccag cgacattgcg atgttttcct 3361 cactgtaata tgattacgtcaaattggtgc gtcattgact tccgcaacct gcgttcatag 3421 ctatcagcta aacatgaaggcaagtacgcg gctatcggaa aatctcaaga acgcataacc 3481 cgatccccgt ccctgcaaacggaacaaggc aaatgacagg cattaccgtt tcagattccg 3541 ctgccaggcg gatcgccaaaattctcgatt cggagccggg aaagaccgcg cttcgcgttt 3601 ctgtcgaagg tggcggctgctccggctttt cctataaata tgacctcgtc gacgcacaga 3661 ccgaggatga catcgtcatcgaaaaactcg gcgccagagt gctgattgat tccatctccg 3721 tgccttatat ggacggctctgaaattgatt tcgtcgatga tctgatgggg caatcattcc 3781 agatccgcaa ccccaatgcgaccgcttcct gcggctgcgg caccagcttc gcgatctgag 3841 cggcgcaaca aaacccgtgatgcaaaaccg gcggccagat ggccgccgtt tttttaacca 3901 tggcaacaag cggacagtttcagactttca ctgaagcaac ggtcgcttcg atgtggtcca 3961 ccagcgcatc ttgcaggccaagccgcccgg caagcatatc cagatagccg cgttcggcgc 4021 ggttatctgg atcgatagccagccgcgatg ccgtataaag ttcaactttc tgctcttccg 4081 tctgcgctgc ggcaaccagcacatcgagat cgacgggttc ggccagttcc cttgcaagga 4141 aggcctcagc ctcgtcgtccagaccggaaa tcttcacctt ttccatgatg cgggcacgtt 4201 cggcatcatc aatataaccatcagccctgg cggcggcgat catggcctga accagcgtca 4261 gcgcgaaact attgctcatcgcgggagaat gcggatggaa gggtgaatcg gccggtggcg 4321 ccggaagaag ctccggctcttttgccaccg gctgttccgc ctcctgcggg gcctgaccgg 4381 acttataatt cttgtaggcaagatagccca atccggctat ggcggcgatg ccgccgacag 4441 ttgctacatt gccagcaagtttgcggcccg ttttcgtgcc aaaaatggct gcggctatgg

TABLE 7 cading sequence af gale-like cading sequence af B. melitensis16M (SEQ ID NO:28). atgacaattcttgtaacaggtggtgctggctatatcggctcccacacgtgtgtgcagttgatcgaggcaggccatgaagtggttgtggtcgataatttcgacaacagccatcctgaggcactgcatcggattgaaaagatcacgggccgcgcgccgcgccgcgaaccgggcgatattcgcgatcgcgcccttatggaacagatgatcaaacgccataaatgcactgcggttatccattttgccgggctgaaggccgtgggtgaatcgagcgaaaagccgctgctctattatgattgcaatgtgctgggcacacttcggcttctgcaggccatggaagcgacaggcgtgaagaagctcgttttcagctcttcggccaccgtctatggcgacccggataaactgccgatcaccgaagatcagcccctttcggccaccaatccctatggccggaccaagcttgtcatcgaagacatgctgcgcgacctttataacagtgacaatagctgggcgattgcgattctgcgctatttcaatcctgtcggcgctcatgaaagcgggcttatcggtgaagacccgaagggtattcccaacaatctgatgcccattattgctcaggtcgcaactggacgacgcgaaaagctgaacatctggggcaacgactatccgacaccggatggcaccggcgtacgcgactatatccatgtcaacgatctggctgccgggcacctcaaggccctgaaaaagctggataagcccaagtgcttcgccgtcaatcttggaacggggcagggctatagtgttcttgatgtgatcaaggcgtttgaacatgtctccaatcgcgagatcaaatatgagattgcgccgcgccgtcccggcgatgttgccgaatgctatgccgatcccggctttgcaaagaaatttctgggctggtcggctgagaaaaacctgcgtgaaatgtgtcaggacatgtggaactggcaatcgaaaaatccgaacggct acgaataa

Although the description herein contains many specific examples anddescriptions, these should not be construed as limiting the scope of theinvention but as merely providing illustrations of some of the presentlypreferred embodiments of the invention. For example, thus the scope ofthe invention should be determined by the appended claims and theirequivalents, rather than by the examples given.

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1. An attenuated strain of Brucella in which there is a mutation whichfunctionally inactivates or prevents expression of at least one of thegalE and having at least 85% nucleotide sequence identity to SEQ IDNO:28, the gene encoding lytic murein transglycosylase and having atleast 85% nucleotide sequence identity to nucleotides 7908-10817 of SEQID NO:26, β-hexosaminidase and having at least 85% nucleotide sequenceidentity with nucleotides 6688-7740 of SEQ ID NO:26, or a gene encodingdeoxyguanosinetriphosphate triphosphohydrolase and having at least 85%nucleotide sequence identity with nucleotides 2138-3346 of SEQ ID NO:27.2. The attenuated strain of claim 1, wherein said mutation is in aBrucella melitensis 16M genetic background and wherein said a mutationwhich functionally inactivates the galE gene of BMEI1090, lytic mureintransglycosylase gene of BME11088 and β-hexosaminidase of BMEI1087 or agene encoding deoxyguanosinetriphosphate triphosphohydrolase and havingat least 85% nucleotide sequence identity with nucleotides 2138-3346 ofSEQ ID NO:27.
 3. The attenuated strain of claim 1, wherein said strainis a Brucella melitensis in which there is a mutation which functionallyinactivates ORF BME10971 or its counterpart in a species of Brucellaother than B. melitensis, the ORF BME11090 or its counterpart in aspecies of Brucella other than B. melitensis, and the operon comprisingORFs BME11090, 1089, 1088 and 1087 or a counterpart operon in a speciesof Brucella other than B. melitensis.
 4. The attenuated strain ofBrucella of claim 1, wherein said mutation is a polar insertionmutation.
 5. The attenuated strain of Brucella of claim 1, wherein saidBrucella is Brucella melitensis.
 6. The attenuated mutant strain ofBrucella of claim 1, wherein said Brucella is Brucella abortus.
 7. Theattenuated strain of Brucella melitensis of claim 3, wherein saidattenuated mutant is GRO24 or GRO26.
 8. The attenuated strain ofBrucella of claim 1 in which there is a deletion in the gene encodingdeoxyguanosine triphosphate hydrolase.
 9. The attenuated strain of claim1, wherein said mutant strain expresses a listeriolysin O gene fromListeria monocytogenes.
 10. An immunogenic composition comprising livecells of at least one attenuated mutant strain of the Brucella of claim1 and a pharmaceutically acceptable carrier.
 11. The immunogeniccomposition of claim 10, wherein said attenuated mutant strain ofBrucella is a Brucella melitensis.
 12. The immunogenic composition ofclaim 11, wherein said attenuated mutant is GR024 or GR026.
 13. Theimmunogenic composition of claim 10 wherein said Brucella is Brucellaabortus.
 14. A method of protecting a human or animal against infectionby administering an effective amount of the immunogenic composition ofclaim
 10. 15. The method of claim 14, wherein said immunogeniccomposition comprises at least one attenuated mutant strain of Brucellamelitensis.
 16. The method of claim 13, wherein said attenuated mutantstrain is GR024 or GR026.
 17. The method of claim 14, wherein saidimmunogenic composition comprises at least one attenuated mutant strainof Brucella abortus.
 18. A method of identifying epitopic peptides of B.melitensis comprising the steps of: (a) infecting macrophage cells inculture with B. melitensis; (b) culturing the macrophage cells infectedwith B. melitensis; (c) collecting MHC class I-peptide complexes fromthe cells cultured in step (b); (d) eluting the peptides from thecollected complexes of step (c); and (e) characterizing the peptideseluted in step (d).
 19. An epitopic peptide identified by the method ofclaim 18, wherein said peptide is derived from the HdeA protein of B.melitensis.